Gap junctions play dynamic roles in cellular processes, however, there is a fundamental knowledge gap in understanding how gap junction proteins, the connexins, are regulated and gated based on a structure that has rigid and flexible domains. Connexin expression and function are highly regulated and the sequence of each isoform imparts specificity (permselectivity) to which molecules pass through the pore. The connexin hexamer (connexon or hemichannel) have three domains defined by the lipid bilayer. Two hemichannels pair at their extracellular domains to form an intercellular channel. The conserved transmembrane and extracellular domains are fairly rigid while the cytoplasmic domain is flexible. The sequence variability in the cytoplasmic domains, particularly in the C-terminus, allows for binding of partner proteins unique to each isoform. Within the context of this compartmentalized structure, our central hypothesis is that the monomer is tightly packed in its rigid domains, but flexibility in the cytoplasmic domains permit supra-molecular complexes to be formed in cells as well as binding of proteins controlling phosphorylation and gating. Connexin-opathies, hereditary human diseases, are often caused by mutations that often disrupt packing or partner interactions. For example, Cx26 mutations account for ~1/2 of cases of pre-lingual non-syndromic deafness in Caucasian populations but cases are found in populations across all continents. The proposed studies explore this hypothesis with three specific aims. (1) To investigate the stability of the transmembrane region of the Cx26 hexamer using mutations known to cause heredity deafness. These experiments will be correlated with ones probing channel function and structure. (2) To determine the 3D structure by cryo-electron microscopy (cryo-EM) and single particle reconstruction of Cx50 hemichannels. Cx50 intercellular channels serve critical functions in lens and its dysfunction leads to cataracts. It has extensive less ordered cytoplasmic domains typically not resolvable by crystallography. In this aim, single particle reconstruction is the best technique to obtain a structure of the large, full-length Cx50 hemichannel. (3) To create electron tomographic volumes of genetically labeled Cx43 intercellular channels and cytoskeletal and scaffolding proteins in situ to better understand the cytoplasmic architecture interacting with a gap junction. Cx43 contains binding domains for cytoskeletal components and the scaffolding protein, ZO-1. It is widespread through most organ systems with particularly important roles in vasculature and heart. The long-term goal is to obtain a more complete depiction of full-length connexins at the highest resolution obtainable. The approach is innovative because it uses a multi-resolution imaging strategy coordinated with biochemical and functional analyses of channels and hemichannels. The proposed research is significant because results will be useful in defining better drugs and other therapeutics that potentially ameliorate connexin-related diseases.